DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werner's syndrome and others.Cruciform structures are targets for many architectural and regulatory proteins, such as histones H1 and H5, topoisomerase IIβ, HMG proteins, HU, p53, the proto-oncogene protein DEK and others. A number of DNA-binding proteins, such as the HMGB-box family members, Rad54, BRCA1 protein, as well as PARP-1 polymerase, possess weak sequence specific DNA binding yet bind preferentially to cruciform structures. Some of these proteins are, in fact, capable of inducing the formation of cruciform structures upon DNA binding. In this article, we review the protein families that are involved in interacting with and regulating cruciform structures, including (a) the junction-resolving enzymes, (b) DNA repair proteins and transcription factors, (c) proteins involved in replication and (d) chromatin-associated proteins. The prevalence of cruciform structures and their roles in protein interactions, epigenetic regulation and the maintenance of cell homeostasis are also discussed.
- MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA chemie metabolismus ultrastruktura MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- molekulární sekvence - údaje MeSH
- regulace genové exprese MeSH
- replikace DNA MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
p53 is one of the most important tumor suppressors which responds to DNA damage by binding to DNA and regulating the transcription of genes involved in cell cycle arrest, apoptosis, or senescence. As it was shown previously, p53 binding to DNA is strongly influenced by DNA topology. DNA supercoiling is fundamentally important for a wide range of biological processes including DNA transcription, replication, recombination, control of gene expression and genome organization. In this study, we investigated the cruciform structures formation of various inverted repeats in p53-responsive sequences from p21, RGC, mdm2 and GADD45 promoters under negative superhelical stress, and analyzed the effects of these DNA topology changes on p53-DNA binding. We demonstrated using three different methods (gel retardation analyses, ELISA and magnetic immunoprecipitation assay) that the p53 protein binds preferentially to negatively supercoiled plasmid DNAs with p53-responsive sequence presented as a cruciform structure. Not only the appearance of the cruciform structures within naked supercoiled DNA, but also the potential of the binding sites for adopting the non-B structures can contribute to a more favorable p53-DNA complex. Copyright 2009 Elsevier Inc. All rights reserved.
- MeSH
- buněčné linie MeSH
- ELISA MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika MeSH
- intracelulární signální peptidy a proteiny genetika MeSH
- jednovláknová DNA chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- obrácené repetice MeSH
- plazmidy chemie metabolismus MeSH
- protoonkogenní proteiny c-mdm2 genetika MeSH
- regulace genové exprese MeSH
- retardační test MeSH
- sekvence nukleotidů MeSH
- superhelikální DNA chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.
- MeSH
- akridiny chemie farmakologie MeSH
- chlorochin chemie farmakologie MeSH
- doxorubicin chemie farmakologie MeSH
- interkalátory chemie farmakologie MeSH
- kompetitivní vazba MeSH
- konformace nukleové kyseliny účinky léků MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie metabolismus MeSH
- superhelikální DNA chemie účinky léků metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BRCA1 is a multifunctional tumor suppressor protein with implications in regulating processes such as cell cycle, transcription, DNA repair, and chromatin remodeling. The function of BRCA1 likely involves interactions with a vast number of proteins and likewise DNA. To this date there is only fragmentary evidence about BRCA1 binding to DNA. In this study, we provide detailed analyses of various BRCA1 protein constructs binding to linear and supercoiled (sc) DNAs. We demonstrate that the central region of human BRCA1 binds strongly to negatively sc plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of sc DNA in agarose gels. At relatively low BRCA1:DNA ratios, binding of BRCA1 to sc DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of BRCA1, the original mobility of the sc DNA is recovered. BRCA1 proteins at higher concentrations also bind to the same DNA but in linear state, leading to formation of a smeared retarded band. Our experiments not only demonstrate a preference for BRCA1 binding to sc DNA, but also show that the central region may contain at least two efficient DNA binding domains with strong affinity for sc DNA. The biological implications of the novel DNA binding activities of BRCA1 are discussed.
The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.
- MeSH
- biologické modely MeSH
- DNA-topoisomerasy I metabolismus MeSH
- DNA fyziologie chemie MeSH
- financování organizované MeSH
- konformace nukleové kyseliny MeSH
- kultivované buňky MeSH
- lidé MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- repetitivní sekvence nukleových kyselin MeSH
- responzivní elementy MeSH
- Spodoptera MeSH
- superhelikální DNA fyziologie chemie MeSH
- tranzitní teplota MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
One of the most important functions of the tumor suppressor p53 protein is its sequence-specific binding to DNA. Using a competition assay on agarose gels we found that the p53 consensus sequences in longer DNA fragments are better targets than the same sequences in shorter DNAs. Semi-quantitative evaluation of the competition experiments showed a correlation between the relative p53-DNA binding and the DNA lengths. Our results are consistent with a model of the p53-DNA interactions involving one-dimensional migration of the p53 protein along the DNA for distances of about 1000 bp while searching for its target sites. Positioning of the p53 target in the DNA fragment did not substantially affect the apparent p53-DNA binding, suggesting that p53 can slide along the DNA in a bi-directional manner. In contrast to full-length p53, the isolated core domain did not show any significant correlation between sequence-specific DNA binding and fragment length.
- MeSH
- časové faktory MeSH
- DNA genetika chemie MeSH
- elektroforéza v agarovém gelu MeSH
- financování organizované MeSH
- geny p53 MeSH
- kompetitivní vazba MeSH
- lidé MeSH
- nádorový supresorový protein p53 genetika MeSH
- plazmidy metabolismus MeSH
- rekombinantní proteiny chemie MeSH
- restrikční enzymy metabolismus MeSH
- statistické modely MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
